Automating NMR Data Collection and Analysis in

Drug Discovery

Steven Hollis, Paul Krolikowski and Chris Wilde Amgen, Inc.

March 21, 2017

Automating Open-Access NMR

• ACD/Labs’ automation solution is used to process and distribute all data in Open-Access NMR at two sites.

• NMR Visor - Custom sample-submittal and tracking softwarefrom Protasis provides real time scheduling and tracking of all NMR submissions over the network from the chemist’s computer.

- Integrates with AS processing system and Bruker Iconnmr.- Structures can be added with sample submission to take . advantage of the ACD/Lab’s NMR processing tools.

- Auto-processing - peak-picking, scaling, integration and plotting .. all are improved when the structure is included with the data.

NMR Processing with ACD/Spectrus

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ACD/Spectrus Processor in Open-Access NMR

• ACD/Labs Automated Solution - provides reliable and reproducible processed-data output directly to the chemists.

• Saves Time - removes the need for repeated manual data processing and plotting.

• Searchable Database - individual database constructed for each chemist for viewing in ACD/Spectrus Processor.

• NMR Visor - includes structures as an option at the time of sample submission in Open-Access NMR.

• Processed-data output is the same as that provided by the High Throughput Purification process.

• Uniform platform for sample tracking and data management. NMR Statistics can be queried using Spotfire tools.

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NMR Visor – a Web Application for Open-Access NMR

NMR VisorScheduling Tool

Data processing uses existing HTPP/Singleton workflow

Runs NMR

Logs in Samplewith Structure

ACD/LabsAutomation

Open-Access NMR Network

Chemist

Reportsto e-LN

Spectrometer

Processing Done by ACD• Integrated NMR output• Creates pdf reports• Structures included• NMR database created• Can search by structure

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NMR Visor: Open-Access Submission and Tracking Tool

Can draw or attach structures, track projects, and more…

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NMR Visor Architecture – Web API Server

Chemist’s Web Access Application DB Server

– WebAPI• REST Architecture• Microsoft C# Controllers

– Oracle SQL Standard Edition• Local or remote• Consistency constraints• Custom Fields

– Active Directory • Secure LDAP Authentication• Windows Authentication

WebAPIC# RESTController

SQLsamples

LDAPdirectory

FileServer

spectra

CloudVPN

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NMR Visor Architecture - Instrument Controller

Real-time scheduler– Custom Priorities– Overnight Samples– Experiment Chains

Windows Application– Client runs on NMR

(with Topspin)– Network Interface– Connects to DB– Shared Drop Folder

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NMR Visor Starts by Showing Spectrometer Status

Submit a sample

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Login Windows Domain

Authenticate against Amgen active directory

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Select an NMR Spectrometer

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Add Sample ID and Choose Experiment Details with Dropdown Menus

The Project field will automatically populate with the last Project used.

The Experiment field will automatically populate with the last experiment that you entered if it’s on the same spectrometer.

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Add Structure with Structure Plugin Tool

Draw freehand, Drag ‘n Drop or

Copy/PasteFrom ChemDraw or ELN

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Can Add More Experiments or Additional Samples

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Submit Sample to Send Request to the NMR

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Put Sample in Holder and Confirm when Ready

After submitting, the samples are placed in the ‘Pending’ state

Chemist must place the sample in the holder on the instrument

Confirm sample on the web page after placing in auto-sampler

Can be done from your lab bench or office

15Click here

Monitor the Sample Queue from Your Desk

• Sample placed in queue - waiting to be run on the instrument• Can view all samples in queue on each instrument

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Review Completed Samples

Displays samples that have completed within the last 7 days

If a user is logged in, list is filtered by that user

Can be filtered by site, spectrometer, chemist, date, etc.

Shows progress of completed or failed samples

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Raw data + structure + metadata copied to a Sweep Directory after acquisition

Processed-data is generated with structure attached by using sdf file generated . by NMR Visor during sample submission process

• ACD/Labs’ auto-processing tries to match integrals to the structure in the processed output (esp, pdf) - this gives better integral values

ACD/Labs Automation

\as_data

NMRData

Acquired

Structure Attachment

Raw Data

NMR Data Processing Flow Diagram

…\processed-dataOutput files

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ACD/Labs Auto-Processing for NMR Samples

ACD/Labs Auto-Processing - raw and processed-data output files are distributed to network-share folder for all samples are run in Open-Access and HTPP

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NMRSpectrum Acquired

2. …\jcamp - in dx or jdx format

1. …\data - raw data in Bruker format

3. …\processed-data – AS output

Open-Access Data Flow

processed-data - contains output files created by ACD/Labs Automation

esp ACD/Labs processed data files in esp formatopen-access-database Open-Access database files in nd9 formatplate-database ACD/Labs plate database files in nd9 formatreports Processed spectra in pdf format structures Chemdraw cdx files - will auto-attach to spectra

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Example NMR Report as pdf

Data summary for notebook injournal format

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Four Page Report – Expansions pages 2-3

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Page 4 - Contains NMR Summary

NMR reports are deposited in the chemist’s reports folder and a copy is sent to the inbox of their Electronic Notebook.

Inspection of the pdf report can often be a suitable for final review without the need for spectrum reprocessing.

The HTPP process also distributes LC/MS data generated for all high throughput purification samples. This processed-data is also sent directly to each users directory on LC/MS file server—accessible for reprocessing in ACD/Spectrus Processor.

Improvements in Open Access NMR

Structures are captured during sample submission so they are available for downstream processing and reporting.

Structural graphics add visual information to improve the final report, helping chemists to confirm and interpret spectra and prevent errors.

ACD/Labs software has proven to be a versatile and reliable tool for processing NMR spectra in automation - using both flow and tube-based NMR techniques.

SQLsamples

An integrated NMR sample history database is fully integrated into the sample management system to improve automation and provide NMR use statistics.

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Making Life Easier for Our Chemists

Chemists can submit crude reaction mixtures directly to the separations group for automated purification and LC/MS/NMR characterization

Can submit up to 250 mg/sample dissolved in up to 2.5 mL of DMSODesired component should be > 50% purity by initial HPLC screenCan submit single samples or entire libraries of up to 24 compounds

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Automating NMR Data Collection and Analysis for HTPP and Sample Registration at Amgen

HTPP Process is an automated mass-directed purification procedure that aids chemists in their efforts to design and synthesize new therapeutic agents.

Chemists employ parallel synthesis to make focused libraries which are purified and characterized using LC-MS and 1H NMR using Flow-NMR techniques.

Purified materials are provided in Sample-Bank vials and processed LC/MS/NMR data are distributed to chemists in electronic format for e-Notebook records.

Design Synthesis ScreeningPurification Characterization Registration

HTPP/Singleton Purification Service DAS Separations

Med ChemD SP C

R

S Biology

Crude reaction mixturesare submitted

MS-Directed

Purification

1. Product Vial2. Solubility Vial3. NMR Plate4. LC/MS QC Plate

Output

General Procedure for the HTPP Process

Sample collection is triggered by Mass Spec and the selected product is dried, weighed and formatted into Sample Bank and Solubility vials and two 96-well plates are prepared in automation using a Tecan Evo robot.

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Mass Directed PurificationUV

MS

Purification Sample Drying Sample Reformatting

Genovac Tecan EvoWaters LC-MS

2 Vials per Sample

LC-MS QC Plate

NMR Plate0.5 µmol / well

2 Plates per Library

Commercial flow cryoprobe system using a 600 MHz Bruker with a 30 µL flow-cell insert in TCI cryoprobe

Utilizes a Protasis solvent pump, LEAP liquid handler and OM-NMR software

Optimized injection volume is 30 µL, much less sample is needed vs. tubes (600 µL for tubes)

S/N testing shows that this system has ~20X better S/N than previous 500 NMR

Proton and g-HSQCdept spectra run in ~20 minutes on 0.5 µmol samples

Enables Automated Structure Verificationanalysis using ACD Automation Server

600 MHz Cryoflow Probe for HTPP-NMR Samples- Improved Sensitivity and Throughput

NMR Flow Cell

30 µL active volume

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ImprovedStructuralAnalysis

HTPP-NMR and Singleton Plates

Pipeline Pilot is used to download sample metadata for direct NMR submissions. SD file is used to collect and process the NMR data - generated from the mass directed purification process.

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HTPP-NMR and Singleton Plates

Example sdf file obtained from Pipeline Pilot - used directly to initiate the NMR data acquisition. Pipeline Pilot assembles the combined metadata for each plate into a single file.

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HTPP-NMR and Singleton Plates

Protasis software strips out each sample from the plate sdf file one line at a time and sends a “single-sample” sdf file to the NMR spectrometer where the file becomes embedded into the NMR data tree for each sample.

• The individual sdf file, which can also be viewed as a simple formatted text file, is saved in the Bruker NMR data tree in the same location that the raw NMR data file is stored (fid file).

• When the data collection is complete the spectrometer software copies the NMR file to a remote server in a sweep directory named AS_data.

• The appearance of the data file in the sweep directory triggers ACD/Labs automation to process, distribute and archive the data in a searchable NMR database that is distributed to the NMR users.

Example: single-sample sdf file - 117442-03-12.sdf

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Running HTPP/Singleton-NMR in Automation

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NMR is Setup by Shooting a Barcode

ReadsSD File

from Pipeline Pilot

• SD file is imported directly into One-Minute NMR

• Generates sample queue

• Click “Queue Run” to start the NMR for the entire plate

SD File

Click Run

One-Minute NMR

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LC/MS Reports Generated by ACD/Labs

HTPP/Singleton Submissions are processed in automation by ACD/Labs• ACD/Spectrus generates two report files in pdf format for the chemists to attach

to their electronic notebooks: LC/MS report and NMR reports for each sample• An example LC/MS report is shown below

LC/UV at 215 and 254 nm TIC’s MS+ and MS-

N

HO

N

O

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1D NMR Reports Generated by ACD/Spectrus Automation - same as Open-Access

• Both reports are added to chemist’s e-LN• Stored on shared processed-data file system

on the network

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For Internal Use Only. Amgen Confidential. 34

Computer Assisted Structure Verification

Improving the Information Content of HTPP- NMR The 600 MHz Cryoflow probe for HTPP-NMR samples provides a

large sensitivity gain (20X S/N) and improved information content benefits the structural verification process. This hardware upgrade:

– Allows combined 1D and 2D experiments in similar time frame– Reduces the chance of placing incorrect samples in Sample Bank– Reduce false readouts during screening and follow-up chemistry– Prevents wasted time and effort in re-evaluating chemistry/SAR

ACD/Labs’ Automated Structure Verification can be implemented using 1H and g-HSQCdept (1H-13C) spectra obtained from standard HTPP-Singleton plates coming from the mass-directed purification

Cryo-Flow Probe NMR systems have been installed in both AMA and ATO for use in the HTPP/Singleton-NMR ASV deployment

ACD/Labs ASV - Key Capabilities

Automated Structure Verification using 1H/HSQC data

– Provides easy to understand ASV reports with explanations, number of CH, CH2, CH3 groups, total proton count, match factor, and color coded structures for quick review.

– Users have the software tools (Spectrus Processor) to reviewthe LC/MS/NMR results for added verification

Increases chemist’s efficacy and productivity – Supports the current chemists’ workflow– Reduce the risk of making wrong submissions to Sample Bank– Saves potential re-synthesis efforts and reduces potential

errors in SAR elaborations

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NMR Data Flow for ASV Processing

as_data - triggers ACD/Labs ASV processing

ASV output files

Data sent to sweep folder (same process)

processed-dataesp - standard 1H esp files without assignments

- add HSQCdept esp files without assignments

reports - keep 1H standard 4 page report files in pdf format - add ASV report 1H/HSQC w/ assignments + Match Factor

spectrus - new ASV output file for each 1H/HSQC dataset

database - keep spectral database in nd9 format for 1H only

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ASV: Automated Structure Verification

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1D and 2D HSQCdept spectra are collected in approximately 20 min Cryoprobe technology yields high quality data which facilitates

automated structure analysis using both 1H and 13C data Provides the chemist with a detailed view of the molecule in a user

friendly format - click through plate shows match of signals to structure

Utilizes ACD/Labs ASV software for 1H, 13C, chemical shift predictions

= good structure match

= needs to be checked

= problem detected

Better Tools for Verification

Cryoprobe Flow-NMR Technology Dramatically Improves NMR Sensitivity

Match Factor (MF)

Match Factor Calculated based on both 1H and 13C assignments from the1D and 2D experiments

Click on well

.

MF = 0.84

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HQSCdept Pulse Sequence- with improved sensitivity

• Phase-sensitive gradient-2D multiplicity edited HSQC using PEP and inversion, with refocusing and matched sweep adiabatic pulses, and gradients in back-inept element

Adiabatic 13C decoupling

Phase behavior of C-H signals vs. Intensity

Editing delay d21=1/2J for 1J(C-H) = 145 Hz gives:

• CH Positive• CH3 Positive• CH2 Negative

DEPT phase depends on J(C-H) coupling

CH2

CH3CH

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dmso-d6

N

OCH3

N

OH 23

45

67

8

9

10

11

12

1415

18

19 21

24

CH2

CH2

CH2

CH2

CH2

10

24

OH

11

915

85

18

1921

14

7

4

3*

*

water

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How to Interpret HSQCdept Spectra: 1H-13C Correlation Plots

2D display shows the 1H spectrum along the X-axis and the 13C spectrum along the Y-axis (this is a one-bond H-C correlation plot).

The cross peaks appear where a given proton is connected to carbon and the Chemical Shifts of each given by the X, Y-axis values.

The cross peaks is color coded, Blue = Positive, Red = Negative and this indicates the number of protons attached to each carbon. CH3 and CH signals are Blue (up) and CH2 signals are Red (down). Good for picking out complex CH2 signals.

No cross peak for a proton signal means the proton is not attached to a carbon (e.g. OH, NH, etc.)

13C

1H Chemical Shift

ChemicalShift

13C axis

1H-axis

Quinidinein

dmso-d6

OH has nocross-peak

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Assigned 1D and 2D NMR spectra can be reviewed in interactive Spectrus Processor window Tables of assignments with atoms are color coded by the goodness of fit: green, yellow, red

Better Tools for Structure ID

ASV: Cryoprobe Technology Dramatically Improves Sensitivity

Automated Structure Verification 2D Data Display

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2D NMR HSQCdept spectrum gives a clear view of assignments - aids with overlapping signals ASV reports are automatically generated and sent to the chemists' reports folder on server

Interactive tools for 2D data display

Example Automated Structure Verification Report

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ASV reports (in pdf format) are automatically generated and sent to the chemists' reports folder Processed-data files for 1H/HSQC data created for interactive review using ACD/Spectrus Processor Database files are generated for each sample submitted for HTPP/Singleton NMR analysis

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Automated Structure Verification Report – Page 5

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ASV reports also show HSQC assignment data to aid the chemist in the structure verification process

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Assignment Summary on last page of report

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Automated Structure Verification will save time and provide added value to the chemists in their quest to develop new therapeutics for Amgen

The ASV analysis will improve the decision making process regarding structure verification - reduce errors in sample submission process

Resulting NMR registration database will facilitate data review for verifying the structural integrity of molecules of interest when new leads are identified in HTS/QC process

Should reduce structural errors in samples being submitted to Sample Bank

Can be used to check integrity of incoming materials purchased from outside vendors/contractors

Advantages of ASV

ACD/Labs’ automation and ASV tools have provided our chemists with real efficiency improvements in their everyday analytical workflow

Acknowledgements

Dave Strand, Protasis Corporation

Wolfgang Goetzinger, Amgen

Ashley Nichols, Amgen

Grace Bi, Amgen

Larry Miller, Amgen

Peter Grandsard, Amgen

Igor Teslya, ACD/Labs

Dave Adams, ACD/Labs

Carol Preisig, ACD/Labs

Arvin Moser, ACD/Labs

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